1. Field of the Invention
This invention relates to a method for increasing the omega-hydroxylase activity in the yeast Candida tropicalis. This invention also relates to C. tropicalis strains having increased omega-hydroxylase activity and to a method of using these strains for the production of dicarboxylic acids.
2. Description of the Related Art
Aliphatic dioic acids are versatile chemical intermediates useful as raw materials for the preparation of perfumes, polymers, adhesives and macrolid antibiotics. While several chemical routes to the synthesis of long-chain alpha, omega dicarboxylic acids are available, the synthesis is not easy and most methods result in mixtures containing shorter chain lengths. As a result, extensive purification steps are necessary. While it is known that long-chain dioic acids can also be produced by microbial transformation of alkanes, fatty acids or esters thereof, chemical synthesis has remained the preferred route, due to limitations with the current biological approaches.
Several strains of yeast are known to excrete alpha, omega-dicarboxylic acids as a byproduct when cultured on alkanes or fatty acids as the carbon source. In particular, yeast belonging to the Genus Candida, such as C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. maltosa, C. parapsilosis and C. zeylenoides are known to produce such dicarboxylic acids (Agr. Biol. Chem. 35; 2033-2042 (1971)). Also, various strains of C. tropicalis are known to produce dicarboxylic acids ranging in chain lengths from C.sub.11 through C.sub.18 (Okino et al., In BM Lawrence, BD Mookherjee and BJ Willis (eds), Flavors and Fragrances: A World Perspective. Proceedings of the 10.sup.th International Conference of Essential Oils, Flavors and Fragrances, Elsevier Science Publishers BV Amsterdam (1988)), and are the basis of several patents as reviewed by Behler and Schindler, in Aliphatic Hydrocarbons in Biotechnology, H. J. Rehm and G. Reed (eds), Vol. 169, Verlag Chemie, Weinheim (1984).
Studies of the biochemical processes by which yeasts metabolize alkanes and fatty acids have revealed three types of oxidation reactions: .alpha.-oxidation of alkanes to alcohols, omega-oxidation of fatty acids to alpha, omega-dicarboxylic acids and the degradative .beta.-oxidation of fatty acids to CO.sub.2 and water. The first two types of oxidations are catalyzed by microsomal enzymes while the last type takes place in the peroxisomes. In C. tropicalis, the first step in the omega-oxidation pathway is catalyzed by a membrane-bound enzyme complex (omega-hydroxylase complex) comprised of a cytochrome P450 monooxygenase and a NADPH-cytochrome reductase. This hydroxylase complex is responsible for the primary oxidation of the terminal methyl group in alkanes and fatty acids (Gilewicz et al., Can. J. Microbiol., 25, p 201 (1979)). The genes which encode the cytochrome P450 and NADPH reductase components of the complex have been identified as P450ALK and P450RED respectively, and have also been cloned and sequenced (Sanglard and Loper, Gene 76, 121-136 (1989)). Fatty acids are ultimately formed from alkanes after two additional oxidation steps, catalyzed by alcohol oxidase (Kemp et al., Appl. Microbiol. and Biotechnol, 28, p370-374 (1988)) and aldehyde dehydrogenase. The fatty acids can be further oxidized through the same or similar pathway to the corresponding dicarboxylic acid. The omega-oxidation of fatty acids proceeds via the omega-hydroxy fatty acid and its aldehyde derivative, to the corresponding dicarboxylic acid without the requirement for CoA activation. However, both fatty acids and dicarboxylic acids can be degraded, after activation to the corresponding acyl-CoA ester through the .beta.-oxidation pathway in the peroxisomes, leading to chain shortening. In mammalian systems, both fatty acid and dicarboxylic acid products of omega-oxidation are activated to their CoA-esters at equal rates and are substrates for both mitochondrial and peroxisomal .beta.-oxidation (J.Biochem., 102, 225-234 (1987)). In yeast, .beta.-oxidation takes place solely in the peroxisomes (Agr. Biol. Chem., 49, 1821-1828 (1985)).
The production of dicarboxylic acids by fermentation of unsaturated C.sub.14 -C.sub.16 monocarboxylic acids using a strain of the species C. tropicalis is disclosed in U.S. Pat. No. 4,474,882. The unsaturated dicarboxylic acids correspond to the starting materials in the number and position of the double bonds. Similar processes in which other special microorganisms are used are described in U.S. Pat. Nos. 3,975,234 and 4,339,536, in British Patent Specification 1,405,026 and in German Patent Publications 21 64 626, 28 53 847, 29 37 292, 29 51 177, and 21 40 133.
In a copending application, Ser. No. 07/432,091, filed Nov. 11, 1989, now U.S. Pat. No. 5,254,466, a method of producing alpha, omega-dicarboxylic acids in high yields by culturing C. tropicalis strains having disrupted chromosomal POX4A, POX4B and both POX5 genes is disclosed. The POX4 and POX5 gene disruptions effectively block the B-oxidation pathway at its first reaction (which is catalyzed by acyl-CoA oxidase) in a C. tropicalis host strain. The POX4 and POX5 genes encode distinct subunits of long chain acyl-CoA oxidase, which are the peroxisomal polypeptides (PXPs) designated PXP-4 and PXP-5, respectively. The disruption of these genes results in a complete block of the .beta.-oxidation pathway thus allowing enhanced yields of dicarboxylic acid by redirecting the substrate toward the omega-oxidation pathway and also prevents reutilization of the dicarboxylic acid products through the .beta.-oxidation pathway.
The specific productivity (grams of dicarboxylic acid/liter/hr) of substantially pure alpha, omega-dicarboxylic acids should be increased by culturing a C. tropicalis strain in which the chromosomal POX4A, POX4B and both POX5 genes have been disrupted and which also has a greater number of P450 genes than the wild type. The substrate flow in such a strain would be redirected to the omega-oxidation pathway as the result of functional inactivation of the competing .beta.-oxidation pathway by POX gene disruption, while an increase in the amount of rate-limiting omega-hydroxylase through P450 gene amplification should increase the rate of substrate flow through the omega-oxidation pathway.